Homologous recombination is a fundamental event in DNA metabolism. Long recognized for its role in generating genetic diversity, recombination is now known to be crucial for DNA repair and the rescue of stalled replication forks. Defects in these repair mechanisms in higher organisms lead to the accumulation of mutations that eventually result in cancer, and the proposed studies are therefore directly relevant to human disease. We are interested in understanding the underlying mechanisms of recombination at the structural level, and propose to study them in a very simple, well characterized organism, namely bacteriophage T4. T4 is an ideal system for these studies because it relies on recombination-dependent replication or RDR and efficient replication fork progression to generate the required levels of DNA during its infection cycle in Escherichia coli. Seven T4 proteins will be studied, UvsX, UvsY, UvsW, UvsW.1, Dda, gp32 and endonuclease VII. The recombination protein triad UvsX, UvsY and UvsW mediate the core of the homologous recombination reaction and are related to the eukaryotic proteins Rad51, Rad52 and Rad54, respectively. UvsW and Dda are helicases that translocate and/or unwind branched nucleic acid structures and have important roles in recombination and replication fork progression. Defects in helicases such as Bloom and Werner are known to cause cancer in humans, and there is evidence that UvsW and Dda may function very similarly to these molecules. UvsW.1 is a previously unknown T4 protein that we have identified, with a putative role in recombination. gp32 is the T4 single-stranded DNA binding protein that is known to have crucial roles in many aspects of T4 DNA metabolism. Finally, endonuclease VII resolves Holliday Junctions to complete the homologous recombination reaction. The mechanisms of, and interactions between, these seven proteins will be studied at the molecular level by a coordinated approach involving X-ray crystallography to study their structures, in vitro methods to study their individual functions and interactions, and in vivo methods to understand their biological roles. A considerable body of preliminary data has been obtained for this project that includes crystal and NMR structures, important preliminary crystals, purified proteins, demonstrations of biochemical activities, and in vivo function based on analysis of T4 mutants. PUBLIC HEALTH RELEVANCE: This project focuses on a fundamental DNA metabolic event that operates in all life forms, homologous recombination (HR). Traditionally associated with the propagation of genetic diversity, HR is now recognized as a major mechanism by which various forms of DNA damage are accurately and rapidly repaired. Many forms of cancer, notably breast cancer, are associated with defects in the HR machinery. The central events of HR are the pairing of DNA strands, the search for homology and the exchange of homologous DNA segments. Although the proteins that mediate this remarkable process are well characterized, the actual mechanism at the structural level is not well understood. The goals of the project are to study HR in the simple phage T4 system, to understand how the component T4 proteins coordinate the reaction, and to study how HR mediates DNA repair in T4 and in higher organisms. The project encompasses structural, genetics and biochemical techniques working in tandem to study these important biological questions.